Abstract: Many flaviviruses such as Dengue virus (DENV), Zika virus, West Nile virus, and Yellow Fever virus cause significant human diseases. However, no clinically approved antiviral therapy is available for treatment of flavivirus infections. Therefore, the development of vaccines and antiviral agents for prevention and treatment of flavivirus infections is a clear public health priority. The flavivirus envelope (E) protein is a highly conserved and key target for developing therapeutic antibodies and vaccines. Several neutralizing antibodies against the DENV E protein have been identified demonstrated efficacy in preventing DENV infection in animal models. However, these antibodies are not ideal candidates as therapeutics due to Fc-receptor mediated ADE effect. In addition, conventional antibodies have other disadvantages, such as relatively large size, low tissue distribution and penetration, less thermal stability, and high cost of scaled-up production. These limitations restrict their practical application to treatment of DENV infections. In contrast, a single domain, termed VHH (variable domain of camelid heavy-chain-only antibody (HcAb)) or nanobody (Nb) has emerged as an effective drug candidate for various applications, including infectious disease management. VHH is the smallest intact antigen-binding fragment (15 kDa) harboring the full antigen-binding capacity of the original naturally occurring antibody, and has an extended complementary-determining region 3 (CDR3) that allows for penetration of very small molecular structures, such as the canyon on the surface of viral envelope proteins. Compared to conventional antibodies, Nbs have many advantages, including comparable high affinity to targets, high intrinsic stability, high flexibility to reach epitopes normally unreachable by conventional antibodies, ease of production and scale-up, low immunogenicity, good tissue penetration and distribution, ease of cross-linking to form multivalent Nbs, lack of Fc domain to avoid ADE, and less cytotoxicity. The unique features of Nbs make them an attractive potential mode of prevention and treatment for DENV and its associated complications. In this R21 proposal, we will explore the potential of Nbs as novel therapeutics for DENV management. The results from this proposal will establish solid basis in general for nanobodies as novel therapeutics, add the proof-of-principle to develop Nbs as a therapeutic option for DENV management, and identify novel Nbs to neutralize DENV. If successful, novel therapy will be developed to combat DENV infections.